大範圍骨缺陷通常不易痊癒且治療方式複雜，牽涉到許多困難的外科手術。近來，結合病毒基因載體與間葉幹細胞療法發展出之新穎技術，已顯示可協助並加速骨修復。桿狀病毒轉導外源基因表現，雖然有相對於其他病毒載體安全的優勢，但是其最大缺點就是表現時間太短，對於需要長時間表現外源治療性基因應用時，其效果不佳。因此，我們以新建構的FLP/Frt hybrid重組桿狀病毒，媒介較長時間的生長因子表現，以達較佳的骨缺陷修復效果。本研究目的探討藉由FLP/Frt基因延長表現系統之新型重組桿狀病毒轉導同種異體紐西蘭大白兔之間葉幹細胞後是否可以有效地修復大範圍頭蓋骨缺陷。首先我們利用帶有FLP/Frt系統的基因重組桿狀病毒Bac-FCOEE決定轉導間隔時間與轉導病毒劑量，並利用d2EGFP結合FLP/Frt與WPRE分析基因延長表現，之後以 Bac-FCBW(表現第二型骨型態蛋白(BMP2))及Bac-FCVW(表現血管內皮細胞生長因子(VEGF))轉導紐西蘭大白兔之間葉幹細胞，觀察頭蓋骨缺陷修復效果。結果顯示，在病毒劑量同為MOI 100的Bac-FLP和帶有Frt site的重組桿狀病毒在間隔4小時進行連續轉導，其重組效率最佳，且並無明顯抗病毒反應，讓帶有FLP/Frt表現系統的新型桿狀病毒可延長基因表現可達到28天。我們將轉導過Bac-FCBW和Bac-FCVW的BMSCs混合種入PLGA載體並移植入紐西蘭白兔頭蓋骨缺陷(critical-sized calvarial bone defect)以評估加速骨缺陷修復的效果。X-ray與H&E染色結果顯示，mock與Bac-CB/Bac-CV的骨缺陷修復情形並不佳，而Bac-FCBW/Bac-FCVW實驗組在植入12週後，確實可以修復缺陷。

A segmental bone fracture generally necessitates extended and complicated surgical methods related to bone and vessel reconstruction, thus autologous or allogenic bone grafting is usually required.Recently, mesenchymal stem cells (MSCs)-based cell therapy and virus-based gene therapy have converged and have shown great potential in assisting and accelerating bone healing. Baculovirus is a novel vector for gene delivery into stem cells, but it only mediates transient expression. Therefore we developed a long-term system whereby one baculovirus expressed FLP recombinase (Bac-FLP) while the other hybrid baculovirus harbored an Frt flanking transgene cassette to assist and accelerate segmental bone healing. In this study, we explored whether FLP/Frt-hybrid baculovirus vector engineered rabbit MSC are able to assist and accelerate segmental calvarial bone healing. Within the BMSCs transduced with Bac-FLP and Bac-FCOEE, all subsequent transductions were performed at MOI 100/100 at an interval of 4 h and no obvious anti-virus reaction, enabling transgene persistence in episomal form and prolonging the expression to >28 days. BMSCs engineered by the conventional baculovirus transiently expressing BMP2/VEGF (S group) only healed the critical size (8 mm) segmental calvarial bone defects in 20 % of New Zealand White rabbits at 12 weekspost-implantation, whereas BMSCs engineered by the hybrid vectors persistently expressing BMP2/VEGF (L group) healed the critical-size defects in 87 % in 8 weeks. Compared with the S group, the L group can accelerated the healing

Airenne KJ, Hiltunen MO, Turunen MP, Turunen AM, Laitinen OH, Kulomaa MS, Yla-Herttuala S. 2000. Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery. Gene Therap 7(17):1499-504.
Bilello JP, Cable EE, Myers RL, Isom HC. 2003. Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatopytes. Gene Therapy 10(9):733-749.
Boyce FM, Bucher NL. 1996. Baculovirus-mediated gene transfer into mammalian cells. Proceedings of the National Academy of Science 93(6):2348-52.
Bruder SP, Jaiswal N, Haynesworth SE. 1997. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. Journal of Cellular Biochemistry 64(2):278-294.
Bruder SP, Kurth AA, Shea M, Hayes WC, Jaiswal N, Kadiyala S. 1998. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. Journal of Orthopaedic Research 16(2):155-162.
Chan ZR, Lai CW, Lee HP, Chen HC, Hu YC. 2006. Determination of the baculovirus transducing titer in mammalian cells. Biotechnology and Bioengineering 93(3):564-571.
Chen CY, Wu HH, Chen CP, Chern SR, Hwang SM, Huang SF, Lo WH, Chen GY, Hu YC. 2011. Biosafety Assessment of Human Mesenchymal Stem Cells Engineered by Hybrid Baculovirus Vectors. Molecular Pharmacology.
Chen HC, Chang YH, Chuang CK, Lin CY, Sung LY, Wang YH, Hu YC. 2009. The repair of osteochondral defects using baculovirus-mediated gene transfer with de-differentiated chondrocytes in bioreactor culture. Biomaterials 30(4):674-81.
Chen HC, Sung LY, Lo WH, Chuang CK, Wang YH, Lin JL, Hu YC. 2008. Combination of baculovirus-expressed BMP-2 and rotating-shaft bioreactor culture synergistically enhances cartilage formation. Gene Therapy 15(4):309-17.
Chuang CK, Lin KJ, Lin CY, Chang YH, Yen TC, Hwang SM, Sung LY, Chen HC, Hu YC. 2010. Xenotransplantation of Human Mesenchymal Stem Cells into Immunocompetent Rats for Calvarial Bone Repair. Tissue Engineering Part A 16(2):479-488.
Cooper LF, Harris CT, Bruder SP, Kowalski R, Kadiyala S. 2001. Incipient analysis of mesenchymal stem-cell-derived osteogenesis.Journal of Dental Research 80(1):314-20.
Cowan CM, Shi YY, Aalami OO, Chou YF, Mari C, Thomas R, Quarto N, Contag CH, Wu B, Longaker MT. 2004. Adipose-derived adult stromal cells heal critical-size mouse calvarial defects. Nature Biotechnology 22(5):560-567.
Devine SM, Hoffman R. 2000. Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Current Opinion in Hematology 7(6):358-363.
Di Bella C, Farlie P, Penington AJ. 2008. Bone regeneration in a rabbit critical-sized skull defect using autologous adipose-derived cells. Tissue Engineering Part A 14(4):483-490.
Dwarakanath RS, Clark CL, McElroy AK, Spector DH. 2001. The use of recombinant baculoviruses for sustained expression of human cytomegalovirus immediate early proteins in fibroblasts. Virology 284(2):297-307.
Filip S, Mokry J, Hruska I. 2003. Adult stem cells and their importance in cell therapy. Folia Biologica 49(1):9-14.
Friedenstein AJ, Chailakhyan RK, Gerasimov UV. 1987. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 20(3):263-72.
Gamradt SC, Lieberman JR. 2004. Genetic modification of stem cells to enhance bone repair. Annals Biomedical Engineering 32(1):136-47.
Hofmann C, Sandig V, Jennings G, Rudolph M, Schlag P, Strauss M. 1995. Efficient gene transfer into human hepatocytes by baculovirus vectors. Proceedings of the National Academy of Science 92(22):10099-103.
Hsu CS, Ho YC, Wang KC, Hu YC. 2004. Investigation of optimal transduction conditions for baculovirus-mediated gene delivery into mammalian cells. Biotechnology and Bioengineering 88(1):42-51.
Hu Y-C, Yao K, Wu T-Y. 2008. Baculovirus as an expression and/or delivery vehicle for vaccine antigens. Expert Review of Vaccines 7:363-371.
Hu YC. 2005. Baculovirus as a highly efficient expression vector in insect and mammalian cells. Acta Pharmacologica Sinica 26(4):405-416.
Hu YC. 2006. Baculovirus vectors for gene therapy. Insect Viruses: Biotechnological Applications 68:287-+.
Khan Y, Yaszemski MJ, Mikos AG, Laurencin CT. 2008. Tissue engineering of bone: material and matrix considerations. Journal of Bone and Joint Surgery Am 90 Suppl 1:36-42.
Klein R, Ruttkowski B, Knapp E, Salmons B, Gunzburg WH, Hohenadl C. 2006. WPRE-mediated enhancement of gene expression is promoter and cell line specific. Gene 372:153-61.
Kofron MD, Laurencin CT. 2006. Bone tissue engineering by gene delivery. Advanced Drug Delivery Reviews 58(4):555-76.
Kondo S, Takahashi Y, Shiozawa S, Ichise H, Yoshida N, Kanegae Y, Saito I. 2006. Efficient sequential gene regulation via FLP-and Cre-recombinase using adenovirus vector in mammalian cells including mouse ES cells. Microbiology and Immunology 50(10):831-43.
Kost TA, Condreay JP. 2002. Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends in Biotechnology 20(4):173-80.
Lee HP, Chen YL, Shen HC, Lo WH, Hu YC. 2007. Baculovirus transduction of rat articular chondrocytes: roles of cell cycle. Journal of Gene Medicine 9(1):33-43.
Levi B, James AW, Nelson ER, Vistnes D, Wu B, Lee M, Gupta A, Longaker MT. 2010. Human Adipose Derived Stromal Cells Heal Critical Size Mouse Calvarial Defects. PLoS One 5(6):-.
Lin CY, Chang YH, Lin KJ, Yen TC, Tai CL, Chen CY, Lo WH, Hsiao IT, Hu YC. 2010. The healing of critical-sized femoral segmental bone defects in rabbits using baculovirus-engineered mesenchymal stem cells. Biomaterials 31(12):3222-3230.
Lin L, Shen Q, Wei XL, Hou Y, Xue T, Fu X, Duan XN, Yu CL. 2009. Comparison of Osteogenic Potentials of BMP4 Transduced Stem Cells from Autologous Bone Marrow and Fat Tissue in a Rabbit Model of Calvarial Defects. Calcified Tissue International 85(1):55-65.
Lo WH, Hwang SM, Chuang CK, Chen CY, Hu YC. 2009. Development of a hybrid baculoviral vector for sustained transgene expression. Molecular Therapy 17(4):658-66.
Luria EA, Owen ME, Friedenstein AJ, Morris JF, Kuznetsow SA. 1987. Bone formation in organ cultures of bone marrow. Cell and Tissue Reserch 248(2):449-54.
Madry H, Kaul G, Cucchiarini M, Stein U, Zurakowski D, Remberger K, Menger MD, Kohn D, Trippel SB. 2005. Enhanced repair of articular cartilage defects in vivo by transplanted chondrocytes overexpressing insulin-like growth factor I (IGF-I). Gene Therapy 12(15):1171-1179.
Merrihew RV, Clay WC, Condreay JP, Witherspoon SM, Dallas WS, Kost TA. 2001. Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells. Joyrnal of Virology 75(2):903-9.
Noshi T, Yoshikawa T, Dohi Y, Ikeuchi M, Horiuchi K, Ichijima K, Sugimura M, Yonemasu K, Ohgushi H. 2001. Recombinant human bone morphogenetic protein-2 potentiates the in vivo osteogenic ability of marrow/hydroxyapatite composites. Artificial Organs 25(3):201-8.
Partridge KA, Oreffo RO. 2004. Gene delivery in bone tissue engineering: progress and prospects using viral and nonviral strategies. Tissue Engineering 10(1-2):295-307.
Peter SJ, Liang CR, Kim DJ, Widmer MS, Mikos AG. 1998. Osteoblastic phenotype of rat marrow stromal cells cultured in the presence of dexamethasone, beta-glycerolphosphate, and L-ascorbic acid. Journal of Cellular Biochemistry 71(1):55-62.
Phillips JE, Gersbach CA, Garcia AJ. 2007. Virus-based gene therapy strategies for bone regeneration. Biomaterials 28(2):211-29.
Pieroni L, Maione D, La Monica N. 2001. In vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors. Human Gene Therapy 12(8):871-81.
Preston SL, Alison MR, Forbes SJ, Direkze NC, Poulsom R, Wright NA. 2003. The new stem cell biology: something for everyone. Journal of Clinical Pathology-Molecular Pathology 56(2):86-96.
Qin Y, Ji H, Wu Y, Liu H. 2009. Chromosomal instability of murine adipose tissue-derived mesenchymal stem cells in long-term culture and development of cloned embryos. Cloning Stem Cells 11(3):445-52.
Risbud MV, Sittinger M. 2002. Tissue engineering: advances in in vitro cartilage generation. Trends Biotechnol 20(8):351-6.
Sandig V, Hofmann C, Steinert S, Jennings G, Schlag P, Strauss M. 1996. Gene transfer into hepatocytes and human liver tissue by baculovirus vectors. Human Gene Therapy 7(16):1937-45.
Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. 2008. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. Journal of Immunology 180(4):2581-2587.
Shoji I, Aizaki H, Tani H, Ishii K, Chiba T, Saito I, Miyamura T, Matsuura Y. 1997. Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors. Journal of General Virology 78 ( Pt 10):2657-64.
Sung LY, Lo WH, Chiu HY, Chen HC, Chung CK, Lee HP, Hu YC. 2007. Modulation of chondrocyte phenotype via baculovirus-mediated growth factor expression. Biomaterials 28(23):3437-47.
Virk MS, Conduah A, Park SH, Liu N, Sugiyama O, Cuomo A, Kang C, Lieberman JR. 2008. Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone 42(5):921-931.
Wang EA, Rosen V, D'Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, Hewick RM, Kerns KM, LaPan P and others. 1990. Recombinant human bone morphogenetic protein induces bone formation. Proceedings of the National Academy of Science 87(6):2220-4.
Yang XB, Bhatnagar RS, Li S, Oreffo RO. 2004. Biomimetic collagen scaffolds for human bone cell growth and differentiation. Tissue Engineering 10(7-8):1148-59.
Zaminy A, Ragerdi Kashani I, Barbarestani M, Hedayatpour A, Mahmoudi R, Farzaneh Nejad A. 2008. Osteogenic differentiation of rat mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells: melatonin as a differentiation factor. Iranian Biomedical Journal 12(3):133-41.
Zeng J, Du J, Zhao Y, Palanisamy N, Wang S. 2007. Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells. Stem Cells 25(4):1055-61.